Since the first report of a myosin mutation causing a human disease in 1990, over 350 mutations in 6 different sarcomeric myosin heavy chain (MyHC) genes have been associated with 11 different diseases in heart and skeletal muscle. Thus, it seems likely that all 10 genes in this family will ultimately be implicated in human disease. The goal of our renewal application is to understand the molecular and cellular pathogenesis of myosin skeletal myopathies and to test 2 therapeutic interventions. We are among a small number of labs equipped to study myosin-based myopathies with an integrated structural, biophysical, cellular, and organismal approach. Myosin is an asymmetric molecule with a globular motor domain and an ?-helical rod domain that is responsible for assembly of the thick filament. Mutations that cause disease are found throughout the molecule. We propose that myosin diseases arise by multiple mechanisms that are highly interdependent. Aim I will focus on identifying the molecular and cellular determinants of myosin myopathies caused by motor domain mutations. The same mutation in the in the ATP binding pocket of the embryonic, perinatal, and ? MyHC motor domains leads to 3 distinct diseases: Freeman Sheldon Syndrome, Variant Carney Complex and hypertrophic cardiomyopathy, respectively. We will measure the cross bridge cycle of the motors bearing these mutations. We will measure the activation states of multiple signaling pathways. We will probe the involvement of the myosin chaperone, Unc45b, in pathogenesis. We will test small molecule therapeutics in genome edited worm models for these mutations. In Aim II we will determine how distal skeletal myopathies are caused primarily by proline substitutions in the ? myosin rod. We will study these structurally, i cells and in mouse models. A therapeutic intervention of allele- specific RNA silencing will be tested in a mouse model. The goal of Aim III is to understand the roles that MYH7b plays in the mouse. MYH7b is found not only in specialized muscles, where it is sarcomeric, but also in a subset of cells in the brain and in hair cells and neurons in the inner ear. Remarkably, a compound heterozygous mutation in MYH7b has recently been linked to hereditary hearing loss. We will determine how striated muscle myosin functions in cells of the inner ear that have no sarcomeres. We will use genetic inactivation, cell-based investigations and biophysical analysis of hearing loss mutations to answer this question. In summary, this integrated approach to understand myosin myopathies and the contribution of MYH7b to hearing loss will provide new insight into the function and dysfunction of myosins in health and disease. We will explore for the first time the response of a portion of the muscle cell phosphoproteome to mutant myosins and attempt therapeutic interventions in skeletal myopathies.